tbuckley/cabot-search huggingface dataset:
Abstract EC.01: Endocardial Hdac3 Promotes Myocardial Trabeculation Through A MicroRNA Pathway
Authors: Jihyun Jang, Deqiang Li
Journal: Circulation Research (2023)

A key process during early heart development is myocardial trabeculation, which is the formation of bundles of myocardium extending into the chamber. The failure of trabeculation often contributes to many forms of congenital heart defects. Proper trabeculation requires coordination among multiple cell types and signaling communication. For instance, growth signals released from the endocardium are required for trabecular myocardial growth and maturation. However, it is poorly understood how these signaling cascades are initiated and regulated in the endocardium. Recently, our lab demonstrated that epicardial histone deacetylase 3 (HDAC3) is required for myocardial compaction. In this current study, we found that the genetic deletion of Histone deacetylase 3 (Hdac3) in the cardiac endothelial cells in mice resulted in early embryo lethality presenting as a hypotrabeculation cardiac phenotype, suggesting that endocardial Hdac3 deletion disrupted critical signals from the endocardium for trabecular myocardium growth. When cultured with supernatant from Hdac3 knockout (KO) mouse cardiac endothelial cells (MCECs), wild-type (WT) mouse embryonic cardiomyocytes also showed decreased proliferation. Single cell RNA-sequencing identified that transforming growth factor ß3 (Tgfß3) along with their extracellular matrix (ECM) downstream targets was significantly downregulated in Hdac3 deficient endocardial cells. Interestingly, chemical inhibition of deacetylase catalytic activity alone resulted in significant downregulation of Tgfß3 in cultured Hdac3 KO MCECs, suggesting that HDAC3 induces Tgfß3 expression in an enzymatic dependent manner. Mechanistically, we identified that miR-129 was significantly upregulated in Hdac3 KO MCECs and Hdac3 endocardial KO hearts. Overexpression of miR-129 repressed Tgfß3 expression in WT MCECs, whereas knockdown of miR-129 restored Tgfß3 expression in Hdac3 KO MCECs. Our findings reveal a critical signaling pathway in which endocardial HDAC3 promotes trabecular myocardium growth by stimulating Tgfß through repressing miR-129, providing novel insights into elucidating the etiology of congenital heart defects and conceptual strategies to promote myocardial regeneration.
Abstract P1081: Inhibition Of <i>Mir-200c</i> Abrogates Post-infarction LV Remodeling Following A Myocardial Infarct.
Authors: Riley J Leonard, Mason Sweat, Steven Eliason, Kathy Zimmerman, Ariana Batz, William J Kutschke, Robert M Weiss, Brad A Amendt
Journal: Circulation Research (2023)

Ischemic injury and adverse post-infarction myocardial remodeling are major causes of heart failure worldwide. Strategies designed to offset cell death with cardiomyocyte regeneration have, to date, not translated to routine clinical practice. m ir-200c modulates the transcripts of cardiogenic transcription factors (TFs) Tbx5 and Gata4 to promote myocyte maturation. We hypothesized that inhibition of mir-200c activity would result in postnatal myocyte de-differentiation and replication sufficient to ameliorate post-infarction decompensation after ischemic injury. To test this hypothesis, we preformed left coronary ligation on Wild-Type (WT) and transgenic mice expressing an RNA inhibitor against miR-200c (I-200c). Echocardiographic left ventricular (LV) ejection fraction (EF) fell from 88.41 ± 1.08% (WT) and 90% ± 1.42% (I-200c) (p=NS) to 28% ± 11.55% (WT) and 36% ± 9.35% at 1 day post injury (DPI) (p =NS). At 21DPI, LVEF was 27% ± 4.31% (WT) and 64% ± 4.25% (I-200c) [p ≤ 0.0001]. Post-infarction LV chamber dilation was inhibited in I-200c mice (1.136 ± 0.19 v 0.67 ± 0.06 p ≤ 0.004 LV Vol/mass). These results indicated that inhibiting miR-200c following ischemic injury can recover cardiac function and prevent LV dilation. Moreover, trichrome stain showed a decrease in fibrosis 21 DPI (WT: 57% ± 17.46 v I-200c: 12% ± 3.01 p ≤ 0.001). To understand the mechanism causing this phenomenon, we analyzed expression of cardiac progenitor markers Tbx5 and Gata4 . Fold change in expression of both TFs were increased at 21 DPI (WT: 1.02 ± 0.09 v I-200c: 2.86 ± 0.76 p ≤ 0.05; WT: 1.00 ± 0.02 v I-200c: 3.08 ± 0.64 p ≤ 0.05). Additionally, I-200c mice have increased fold change of the cardiac progenitor factor Nkx2.5 (1.01 ± 0.07 v 4.16 ± 1.08 p ≤ 0.05). Tbx5, Gata4, Nkx2.5, in the context of ischemic injury, are thought to perturb post-infarct remodeling and promote CM survival. Conclusions: I-200c treatment abrogates post-infarction LV remodeling and significantly restores LV systolic function, which are associated with increased myocyte expression of cardiogenic progenitor markers. Future studies will determine the importance of myocyte proliferation vs. humoral modulation of surviving myocyte phenotype, toward protection of post-infarction LVs.
Abstract EC.06: Skeletal Muscle Actin Mutation R256H Disrupts Actin Polymerization And Cardiomyocyte Contractility: A Multi-scale Study Of Genetic Dilated Cardiomyopathy
Authors: Ankit Garg, Silvia Jansen, Kory J Lavine, Michael Greenberg
Journal: Circulation Research (2023)

Actin is conserved in all eukaryotic cells with six isoforms in humans. Skeletal muscle actin (ACTA1) is the primarily expressed isoform in skeletal muscle; however, it is also expressed at low levels in the heart. ACTA1 mutations are well known to cause skeletal myopathy, but mechanistic insights are incomplete due to difficulties with generating recombinant protein and limited in vivo modeling. The impact of ACTA1 mutations on cardiac function is controversial. We previously reported a novel missense heterozygous mutation in ACTA1, R256H, associated with dilated cardiomyopathy (DCM) without overt skeletal myopathy in patients. The objective of this study was to define the mechanistic basis by which ACTA1 R256H contributes to DCM. To establish the pathogenicity of ACTA1 R256H, we generated Acta1 R256H mice. Acta1 R256H/R256H mice display perinatal mortality. We also observed reduced survival and gross phenotypes in Acta1 R256H/+ mice over time. To further characterize ACTA1 R256H in human cardiomyocytes, we generated ACTA1 R256H/+ human induced pluripotent stem cells which we differentiated into cardiomyocytes. ACTA1 R256H/+ cardiomyocytes generated less force and displayed aberrant sarcomere structure compared to isogenic controls. To biochemically define the consequences of ACTA1 R256H protein, we developed a novel method to generate recombinant wildtype ACTA1 and ACTA1 R256H. Strikingly, ACTA1 R256H demonstrated severely reduced polymerization kinetics possibly accounting for the observed disruption in sarcomere structure. This finding was further supported by molecular dynamic simulations which identified impaired nucleotide binding in ACTA1 R256H compared to wildtype ACTA1 protein. Together, these findings reveal that a mutation in skeletal muscle actin results in increased mortality, cardiomyocyte hypocontractility, sarcomeric disruption, and impaired actin polymerization. Our work defines a cardiac function for skeletal muscle actin and establishes a multiscale approach to studying cardiomyopathy-associated actin mutations.
Abstract P3145: Enhancing Cardiac Cav1.2 Ion Channel Function By Targeted Deubiquitination
Authors: Sri Karthika Shanmugam, Papiya Choudhury, Henry M Colecraft
Journal: Circulation Research (2023)

Ventricular excitation-contraction coupling relies on precise spatial arrangement of voltage-gated calcium channels CaV1.2 within T-tubular dyadic junctions proximal to ryanodine receptors (RyR2) that gate Ca2+ release from the sarcoplasmic reticulum. Impaired CaV1.2 trafficking is linked to devastating cardiovascular diseases such as arrythmias and cardiac hypertrophy. Ubiquitination is an important regulatory mechanism that controls ion channel surface trafficking and turnover. Emerging evidence hint at a prominent role for CaV1.2 ubiquitination to underlie heart failure disease progression, that suppresses CaV1.2 currents (ICa,L), resulting in harmful compensatory mechanisms. Targeted protein stabilization (TPS) is a burgeoning therapeutic approach for undruggable targets. We hypothesized that harnessing deubiquitinases using TPS can counteract pathological ubiquitination of CaV1.2, overcoming the need for hormonal remodeling and lead to novel treatment. To this end, we sought to develop engineered deubiquitinases (enDUBs) that can increase functional expression of CaV1.2 channels in the heart. The catalytic domain of ubiquitin specific protease 21 (USP21) fused to nb.F3 (F3-USP21) robustly increased CaV1.2 currents reconstituted in HEK293 cells as well as endogenous ICa,L in mammalian ventricular cardiomyocytes. This increase was within the physiological range of the impact of β-adrenergic stimulation of CaV1.2 and did not alter ventricular action potential duration. The mechanism of nbF3-USP21-induced increase in ICa,L is augmented CaV1.2 surface density. Reassuringly, this approach did not adversely interfere with the correct placement of CaV1.2 channels at dyadic junctions as cardiomyocytes expressing nb.F3-USP21 displayed normal colocalization of CaV1.2 with RyR2 at T-tubules. In conclusion, our study provides evidence that targeting deubiquitinases using TPS could be a viable therapeutic approach for rescuing CaV1.2 functional expression and these findings may pave the way for future development of TPS-based therapies for cardiovascular diseases such as arrhythmias and cardiac hypertrophy.
Abstract P2185: Hydrogen Sulfide Donor Inhibits Cisplatin-induced Foam Cell Death In An In Vitro Model Of Atherosclerosis
Authors: Nigeste M Carter, Adolfo G Mauro, Elisa Palladino, Allison L Roden, Eleonora Mezzaroma, Stefano A Toldo, Fadi N Salloum
Journal: Circulation Research (2023)

Introduction: Cisplatin (CPDD) is used to treat various cancers, including testicular, lung, head and neck, and ovarian. CPDD use is associated with long-term cardiovascular morbidity and mortality risk, including acute coronary syndrome (ACS). Pro-inflammatory activation and death of macrophage foam cells (MFC) within the atheroma contribute to plaque destabilization and coronary artery thrombosis. The mechanisms underlying CPDD-induced ACS remain largely obscure. Hydrogen sulfide (H 2 S) has been shown to regulate cell survival and reduce the residual inflammatory risk in the setting of myocardial infarction. Hypothesis: We hypothesize that H 2 S protects MFC following CPDD administration. Methods: Human monocyte THP-1 cells were differentiated into MFC with 150 nM of phorbol 12-myristate 13 acetate (PMA) for 48 hours, followed by 100 μg/mL of OxLDL in serum-free media for 24 hours. Lipid content was assessed by Oil Red O staining (Fig A). A Methyl thiazolyl diphenyl-tetrazolium bromide (MTT) assay was used to assess the cytotoxic effects on MFC subjected to 24-hour treatment with 25 μM CPDD following 30-minute pre-treatment with 40 μM sodium sulfide (Na 2 S, a H 2 S donor) (Fig B). Results: Differentiated cells exposed to OxLDL showed a 25% increase in lipid accumulation compared to untreated cells (Fig A, **p&lt;0.01 vs THP-1). Na 2 S alone showed no effect on cell death. Treatment with CPDD induced ~20% increase in cell death while pre-treatment with Na 2 S reduced MFC death by ~50% (Fig B, **p&lt;0.01 vs OxLDL, *p&lt;0.05 vs OxLDL+CPDD). Conclusion: Our results indicate that CPDD may contribute to plaque destabilization by inducing MFC death, which is attenuated with H 2 S treatment.
Abstract P2058: Mesenchymal Stem Cells Reduce Endothelin-1 Induced Hypertrophy In Hypoplastic Left Heart Syndrome Patients’ Cardiomyocytes
Authors: Xuebin Fu
Journal: Circulation Research (2023)

Introduction: Hypoplastic left heart syndrome (HLHS) is one of the most difficult congenital cardiovascular conditions. There are no proven therapeutic strategies for systemic right ventricular (RV) heart failure till now. Hypothesis: We investigated whether mesenchymal stem cells (MSCs) can ameliorate heart failure (HF) in HLHS patients. Methods &amp; Results: We analyzed the cardiac tissues of patients with HLHS (N = 20) and healthy donors (N = 12). Compared to the controls, the cross-sectional area of cardiomyocytes determined by wheat germ agglutinin staining significantly increased in the HLHS hearts. Furthermore, quantitative RT-PCR (qRT-PCR) confirmed the expression of fetal genes, including atrial natriuretic factor (ANF), natriuretic peptide B (BNP), and myosin heavy chain-β (MHC-β), were significantly upregulated in the HLHS. The results indicated severe pathological hypertrophy in HLHS patients. To investigate our hypothesis, we employed HLHS patients’ iPSCs and differentiated them into cardiomyocytes (iCMs). Differentiated HLHS-iCMs were confirmed by expression of the cardiac markers, cardiac troponin T. In vitro , 10nM Endothelin-1 (ET-1) stimulation successfully induced cardiac hypertrophy confirmed by cell size and fetal gene expression. Interestingly, co-cultured HLHS-iCMs with mesenchymal stem cells (MSCs) in the trans-well plates reduced the ET-1 induced hypertrophy. The results indicated that secretome from MSCs played a critical role in referring to the anti-hypertrophic effects. Compared to the control conditioned media from fibroblasts, GDF-15 was highly expressed in the MSC-conditioned medium. GDF-15 alone also reduced ET-1 induced hypertrophy in the HLHS-iCMs. Conclusions: In HLHS patients, RV hypertrophy is a hallmark of heart failure. MSCs potentially inhibited hypertrophic remodeling in HLHS patient-specific cardiomyocytes through beneficial secretomes, such as GDF-15.
Abstract P1083: Retinoid Regulation Of Cardiac Inflammation
Authors: Ian Gans
Journal: Circulation Research (2023)

Survivors of acute cardiac events (e.g., myocardial infarction, MI) often recover poorly due to pathological remodeling and progressive heart failure. Cell-based therapies hold potential to improve recovery but have had limited success. Our lab has found that infusion of highly proliferative cardiac cell clones from patient biopsies can improve recovery from MI in mice, but with variable efficacy stemming from unknown factors. Because Vitamin A (VA) stores are known to be mobilized to the heart in response to MI, we investigated the effect of VA on patient-derived cardiac cells. Clones were cultured and treated with 500 nanomolar retinoic acid (RA, bioactive metabolite of VA) for 48 hours. Protein was extracted and digested for proteomic analysis using liquid chromatography and mass spectrometry. To investigate interaction of RA with pro-regenerative epidermal growth factor (EGF) family receptor signaling, cells were treated with 50ng/ml recombinant neuregulin (NRG1) alone or in co-treatment with RA. Treatment with RA caused significant increase in protein expression of inflammatory mediators including IL1A/B, ICAM1, prostaglandin synthase, and NFkB2, and less studied targets including differentiation factor latexin (LXN) and orphan G-protein coupled receptor GPRC5A. Expression was validated at the transcript level. NRG1 treatment significantly modulated expression of metabolic regulators carnitine-palmitoyl transferase, phosphofructokinase, and dipeptidyl peptidase but was inhibited by co-treatment with RA. Our data suggest that RA may induce a pro-inflammatory phenotype in proliferative cardiac cells and that RA inhibits pro-regenerative NRG1/EGF receptor signaling, perhaps as part of an emergency inflammatory response to injury. While the biology underlying limited successes of cell therapies to treat heart disease is incompletely understood, managing inflammation is one strategy that has shown benefits in treatment of heart failure and reprogramming cells to therapeutic ends. Further experiments are called for to investigate an inflammatory role of VA during MI in vivo, and to determine how the interaction of VA and NRG1 may regulate proliferative cardiac cell fate and function after injury.
Abstract P2053: Bag5 Plays A Critical Role In The Regulation Of Cellular Homeostasis Through Fine-tuning Of Cellular Protein Quality Control
Authors: Avni Mukker, Shiridhar Kashyap, Manish K Gupta
Journal: Circulation Research (2023)

Background: Cardiac hypertrophy is one of the heart's adaptive responses, which may play a compensatory role in enhancing cardiac function during stress conditions. However, prolonged hypertrophy condition can lead to the progression of cardiac dysfunction and heart failure. Cellular chaperones and co-chaperones play a critical role in maintaining cellular homeostasis by regulating cellular protein quality control. A Bcl2 binding protein BAG5 acts as a co-chaperone to heat shock protein 70 and is known to be upregulated during oxidative stress. A recent study suggests that mutation of the BAG5 gene induces cardiac dysfunction by promoting dilated cardiomyopathy and arrhythmia. However, its role in cardiac hypertrophy is not clear. Method and Results: This study explores the BAG5 function through a protein-protein interaction study by immunoprecipitation and mass spectroscopy. Further, the role of BAG5 in cellular protein quality control and cardiac hypertrophy was tested using cardiomyocytes. Our protein interaction study suggests that BAG5 interacts with several cellular proteins involved in the biological process like Golgi-associated vesicular transport, fatty acid degradation, endoplasmic reticulum-assisted protein folding, chaperone-mediated protein degradation, and deubiquitinating process. Further, we found that knockdown of BAG5 causes induction of cellular hypertrophy, and overexpression of BAG5 reduces PE and ISO-induced cellular hypertrophy. Additionally, we found that BAG5 maintains a balance of cellular protein quality control, and knockdown of BAG5 causes upregulation of cellular autophagy Conclusion: BAG5 plays a critical role in maintaining cellular homeostasis and is protective during cardiac hypertrophy. Additionally, the reduction of BAG5 protein can promote maladaptive autophagy.
Abstract P2051: Fibroblast-specific TGF-b Signaling Promotes Interstitial Fibrosis, Cardiomyocyte Hypertrophy And Cardiac Dysfunction In The Diabetic Heart Through Smad3 Activation
Authors: Izabela Tuleta, Linda Alex, Harikrishnan Venugopal, Claudio Humeres, Silvia C Hernandez, Akihiko Kubota, Anis Hanna, Nikolaos G Frangogiannis
Journal: Circulation Research (2023)

Objectives: Patients with diabetes exhibit a high prevalence of HFpEF associated with interstitial fibrosis and cardiomyocyte hypertrophy. However, the cellular and molecular mechanisms involved in diabetic heart disease remain poorly understood. TGF-bs are potent fibrogenic mediators that are activated in diabetic hearts, and act through a single type 2 receptor, TbRII. We hypothesized that fibroblast-specific actions of TGF-bs may mediate fibrosis and dysfunction in diabetic hearts. Methods and Results: To test the hypothesis, we developed lean and db/db type 2 diabetic TbRII KO mice (FTbRII2KO) using the Col1a2 CreER driver. Fibroblast-specific loss of TGF-b signaling attenuated hypertrophy in db/db mouse hearts. Speckle-tracking echocardiography with strain imaging showed that fibroblast-specific TbRII loss improved systolic and diastolic cardiac function in diabetic animals. In contrast, fibroblast TbRII disruption caused a worsening of both cardiac systolic and diastolic function in lean animals. Attenuated dysfunction in diabetic mice lacking fibroblast TGF-b signaling was associated with reduced collagen fiber thickness and with decreased cardiomyocyte size, suggesting paracrine effects of fibroblasts on cardiomyocytes. To study the downstream cascade responsible for the effects of TGF-b, we generated lean and diabetic mice with fibroblast-specific Smad3 loss. In db/db mice, fibroblast-specific Smad3 loss phenocopied the effects of fibroblast-specific TbRII disruption, suggesting that in diabetic cardiac fibroblasts TGF-b acts through Smad3 signaling. The bioinformatic analyses of transcriptomic profiles of cardiac fibroblasts identified thrombospondin 4 as a candidate mediator of Smad3-dependent diabetes-associated activation. Conclusions: Fibroblast-specific TGF-b actions in diabetes promote cardiac dysfunction through effects on collagen deposition and via paracrine actions on cardiomyocytes. The effects of TGF-b on diabetic cardiac fibroblasts are Smad3-dependent. In contrast to the deleterious effects of fibroblast TGFb/Smad3 signaling in diabetic hearts, TGF-b-activated Smad3-dependent signaling is important to maintain homeostatic function and geometry in normal lean animals.
Abstract P2179: Endothelial-derived Extracellular Vesicles Communicate With Cardiomyocytes To Elicit Cancer Therapy-related Cardiac Dysfunction
Authors: Crizza Ching, Cassandra L Clift, Caroline Kinnear, Dakota Gustafson, Elena Aikawa, Sasha A Singh, Seema Mital, Paaladinesh Thavendiranathan, Jason Fish
Journal: Circulation Research (2023)

Background: Treatment with the anthracycline, epirubicin, can result in cancer-therapy related cardiac dysfunction (CTRCD), a leading cause of non-malignant mortality in cancer survivors. Endothelial cells (ECs) lining the blood vessels are the first point of contact of epirubicin and secrete factors that are pivotal in regulating cardiac function. Of these factors, extracellular vesicles (EVs) are known to mediate intercellular communication. It remains unknown whether and how EC-EVs affect cardiomyocyte function in the context of CTRCD. Hypothesis: We hypothesize that epirubicin exposure leads to altered EC-EV cargo which mediates cardiomyocyte dysfunction. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to the peak and trough concentrations of epirubicin and endothelial function was assessed. EC-EVs were isolated via ultracentrifugation and EV size and concentration were measured through nanoparticle tracking analysis. The protein content of EC-EVs was determined using liquid chromatography with tandem mass spectrometry. Pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were exposed to physiological concentrations of EVs (1x1010 particles/mL) and contractility was assessed in real-time. Results: Epirubicin exposure increased endothelial leak, apoptosis, and adhesion molecule expression. We observed an increase in size and concentration of EC-EVs and proteomic analysis revealed altered EV cargo including reduced cardiomyocyte protective factors, CNP and eNOS in EVs from epirubicin treated HUVEC in comparison to control. Treatment of iPSC-cardiomyocytes with EVs from epirubicin treated HUVECs resulted in reduced contractility compared to EVs from healthy ECs. Cleavage of EC-EV surface proteins via proteinase K abrogated the effects of EC-EVs on iPSC-cardiomyocytes. Cardiomyocyte-specific pathways targeted by EC-EVs from epirubicin- compared to control-treated cells are currently on-going. Conclusion: We demonstrate that epirubicin treatment leads to endothelial dysfunction and altered release of EVs capable of mediating cardiomyocyte dysfunction. Our results show the potential role of EC-EVs in CTRCD pathogenesis and highlight their influence on cardiomyocyte function.